MG53 suppresses NF-κB activation to mitigate age-related heart failure

Figures

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  Figure 1. Failing human hearts and aged mouse hearts show reduced MG53 and increased p65 activation. (A) Western blot of MG53, total p65 (t-p65), and phosphorylated p65 (p-p65) in nonfailing human hearts (NFHs, n = 13) and failing human hearts (FHs, n = 15). GAPDH serves as loading control. (B) Quantification of MG53, t-p65, and p-p65 in NFH and HF tissues. (C) Inverse correlation between MG53 and p-p65 expression in human heart tissues. (D) Left ventricular ejection fraction (LV EF) in young (3 months, n = 15) and aged (24 months, n = 14) mice. (E) Western blot for MG53, p-p65, p65 in young mouse hearts (YH, 3 months, n = 4) and aged (AH, 24 months, n = 4) mouse hearts. (F) Quantification of MG53, t-p65, and p-p65 expression in young and aged mouse hearts. Data are expressed as mean ± SEM. Differences were analyzed for significance by unpaired t test; P values are presented in the individual panels.

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  Figure 2. Systemic administration of rhMG53 mitigates HF in aged mice. (A) Schematic protocol for the 6-week daily administration of rhMG53 in aged mice. (B) Survival curve for mice with systemic administration of rhMG53 (n = 30) or saline control (n = 30); 15 males and 15 females mice per group. (C) Longitudinal echocardiography measurements of aged mice with saline and rhMG53 treatments. (D) Representative pressure-volume loop for the assessment of aged mouse heart function after treatment with saline or rhMG53. (E) Quantitative analysis of the left ventricular EF for the PV-loop measurements for mice treated with saline and rhMG53. Open symbols, male mice; closed symbols, female mice. Data are expressed as mean ± SEM. Differences were analyzed for significance by unpaired t test (C and E); P values are presented in the individual panels.

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  Figure 3. Longitudinal treatment with rhMG53 suppresses activation of the NF-κB signaling pathway and cardiac cell death. (A) Quantitative real-time PCR analysis showing the relative mRNA expression of proinflammatory marker genes Ifng (IFN-γ), Il1b (IL-1β), Il6 (IL-6), and Tnfa (TNF-α), in cardiac muscle collected from young mice (YH, 3 months) and aged mice (AH, 25.5 months) treated with saline or rhMG53 (n = 8 per group). (B and C) Western blot and quantitative analysis of MG53, phosphorylated and total p65, IL-6, TNF-α, and GAPDH (as the loading control) in heart tissues derived from young mice and aged mice treated with saline or rhMG53 (n = 3). (D and E) Representative confocal images and quantitative analysis for the TUNEL staining of heart sections from aged mice treated with rhMG53 (n = 6) or saline (n = 5). Scale bar: 100 μm. Data are expressed as mean ± SEM. P values were calculated using 1-way ANOVA with Tukey’s multiple comparison test (A and C) or an unpaired t test (E) and are presented in the individual panels.

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  Figure 4. Daily administration of rhMG53 regulated the expression of oxidative stress–associated proteins and suppressed age-related ROS generation. (A and B) Western blot and quantitative analysis of catalase, GPX1, NOX4, and GAPDH (as the loading control) in heart tissues derived young mice (3 months) and aged mice treated with saline or rhMG53 (25.5 months). The samples and loading condition were identical to those in Figure 3B. (C–E) Cross sections of mouse left ventricles stained with dihydroethidium (DHE) for quantification of ROS in young mice (C) and aged mice treated with saline (D) or rhMG53 (E) (n = 3). Scale bars: 50 μm. (F) The quantification of the data in C–E. Data are expressed as mean ± SEM. P values were calculated using 1-way ANOVA with Tukey’s multiple comparison test (B and F) and are presented in the individual panels.

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  Figure 5. Repetitive administration of rhMG53 is safe in aged mice. (A) The ratios of heart weight/tibia length (HW/TL), liver weight/body weight (BW), and spleen weight/BW were not significantly changed after 6-week administration of rhMG53 in aged mice versus the saline group. (B) Serum levels of ALT, ALP, and total bilirubin in mice treated with saline or rhMG53. (C) Serum levels of LDL, HDL, and triglycerides in mice treated with saline or rhMG53. (D) Serum levels of LDH and CK in mice treated with saline or rhMG53. Open symbols, male mice; closed symbols, female mice. Data are expressed as mean ± SEM. Differences were analyzed for significance by an unpaired t test (A–D); P values are presented in the individual panels.